Kinases are important cellular enzymes that perform essential cellular functions such as regulating cell division and proliferation, and that appear to play a decisive role in many disease states such as in disease states that are characterized by uncontrolled proliferation and differentiation of cells. These disease states encompass a variety of cell types and maladies such as cancer, atherosclerosis, restenosis and other proliferative disorders.
One of the most important and fundamental processes in biology is the division of cells mediated by the cell cycle. This process ensures the controlled production of subsequent generations of cells with defined biological function. It is a highly regulated phenomenon and responds to a complex set of cellular signals both within the cell and from external sources. A complex network of tumor promoting and suppressing gene products are key components of this cellular signalling process. Over-expression of tumor-promoting components or the subsequent loss of the tumor-suppressing products will lead to unregulated cellular proliferation and the generation of tumors (Pardee, Science 246:603-608, 1989). Cyclin-dependent kinases (CDKs) play a key role in regulating the cell cycle machinery. They are complexes consisting of two components: a catalytic subunit (the kinase) and a regulatory subunit (the cyclin). To date, thirteen kinase subunits (cyclin-dependent kinases (CDKs) 1-13) have been identified in humans along with several regulatory subunits including cyclins (Cyc) A-H, K, L, N, and T, and CDK5, p35, and other proteins. Each kinase subunit can form pair(s) with one or several regulatory subunit partners, and in each case, such pair makes up the active catalytic moiety. Each transition of the cell cycle is regulated by a particular cyclin-dependent kinase complex: G1/S by CDK2/CycE, CDK4/CycD and CDK6/CycD; S/G2 by CDK2/CycA and CDK1/CycA; G2/M by CDK1/CycB (for review see Shapiro, J. Clin. Oncol. 24: 170ff, 2006). The coordinated activity of these kinase complexes guides the individual cells through the replication process and ensures the vitality of each subsequent generation (Sherr, Cell 73:1059-1065, 1993; Draetta, Trends Biochem. Sci. 15:378-382, 1990).
While experiments disrupting the genes encoding all three D-type cyclins, the two E-type cyclins, cyclin D-dependent CDK4 and CDK6, or cyclin E-dependent CDK2 in the mouse germ line showed that none of these genes is strictly essential for cell cycle progression (reviewed in Sherr and Roberts, Genes & Development 18: 2699-2711, 2004), an increasing body of evidence has shown a link between tumor development and cyclin-dependent kinase related malfunctions. Over-expression of the cyclin regulatory proteins and subsequent kinase hyperactivity have been linked to several types of cancers (Sherr C. J., Science 274:1672-1677, 1996; Jiang, Proc. Natl. Acad. Sci. USA 90:9026-9030, 1993; Wang, Nature 343:555-557, 1990). Indeed, human tumor development is commonly associated with alterations in either the CDK proteins themselves or their regulators (Cordon-Cardo C., Am. J. Pathol. 147:545-560, 1995; Karp J. E. and Broder S., Nat. Med. 1: 309-320, 1995; Hall M. et al., Adv. Cancer Res. 68:67-108, 1996). Endogenous, highly specific protein inhibitors of cyclin-dependent kinases were found to have a major effect on cellular proliferation (Kamb A., Curr. Top. Microbiol. Immunol. 227:139-148, 1998; Kamb et al., Science 264:436-440, 1994; Beach, Nature 336:701-704, 1993). These inhibitors include p16INK4 (an inhibitor of CDK4/CycD1), p21CIP1 (a general CDK inhibitor), and p27K1P1 (a specific CDK2/CycE inhibitor). A crystal structure of p27 bound to CDK2/CycA revealed how these proteins effectively inhibit the kinase activity through multiple interactions with the cyclin-dependent kinase complex (Pavletich, Nature 382:325-331, 1996). These protein inhibitors help to regulate the cell cycle through specific interactions with their corresponding cyclin-dependent kinase complexes. Cells deficient in these inhibitors are prone to unregulated growth and tumor formation.
In addition to the CDKs involved primarily in the core process of cell cycle progression (CDKs 1, 2, 4 and 6), other CDKs are responsible for regulating gene expression processes in the course of cell cycle progression. Specifically, CDK7 and CDK9 are known to phosphorylate the C-terminal domain of RNA Polymerase II and thereby drive the expression of anti-apoptotic proteins, D-type cyclins, and pro-angiogenesis factors (like hypoxia-induced VEGF). Therefore, also these so-called regulatory CDKs are attractive targets for therapeutic intervention (see Shapiro, J. Clin. Oncol. 24:1770ff, 2006).
Due to the clear connection between pharmacological CDK inhibition and cell cycle regulation, the potential use of CDK inhibitors in oncology was identified early on, and many small-molecule inhibitors belonging to different structural classes were identified (e.g., staurosporins, flavones (e.g., flavopiridol (see Shapiro, J. Clin. Oncol. 24:170ff, 2006)), purines (e.g., purvalanol; roscovitine (see Bach et al., J. Biol. Chem. 280: 31208ff, 1995)), pyrido[2,3-d]pyrimidinones, oxindoles, paullones, indenopyrazoles, anilinoquinazoline, aminothiazoles or diaryl ureas, and the first molecules are now undergoing clinical evaluation (for reviews see: Sausville, Trends Mol. Med. 8: S32-S37, 2002; Huwe et al., Angew Chem Int Ed Engl. 42: 2122-38, 2003; Fischer, Cell Cycle 3: 742-6, 2004; Fischer and Gianella-Borradori, Expert Opin Investig Drugs 14: 457-77, 2005).
Based on the current understanding of the biochemical roles of the CDKs 1, 2, 4 and 6, growth arrest can be expected in cells treated with inhibitors of these enzymes. Direct inhibition of CDK4/CycD and/or CDK6/CycD should arrest cells in G1 (Baughn et al., Cancer Res. 66: 7661-7, 2006). Moreover, indirect inhibition of CDK1 and CDK4 via down-regulation of the corresponding cyclin partners through inhibiting the CDK-activating kinases CDK7 and/or CDK9, should also arrest cells in G1 (Whittaker et al., Cancer Res. 2004, 64, 262-72; Mateyak et al., Mol Cell Biol. 1999, 19, 4672-83). In principle, inhibition of CDK2 activity should result in arrest of cells in G1, however, it has been shown genetically in colon cancer cells that this block could be by-passed by CDK4 activity (Tetsu & McCormick, Cancer Cell 3: 233-45, 2003) Inhibition of CDK1/CycB should block exit from mitosis through inhibiting phosphorylation of components of the anaphase-promoting complex (Golan et al., J Biol. Chem. 277: 15552-7, 2002; Listovsky et al., Exp Cell Res. 255: 184-91, 2000), and it could also result in apoptosis through inhibiting the phosphorylation of Survivin (O'Connor et al., Proc Natl Acad Sci USA. 97: 13103-7, 2000).
In light of the above considerations a molecule with potent and balanced CDK inhibitory activities, particularly against CDK1, CDK2, CDK4, CDK6, CDK7 and CDK9, would be expected to be a promising candidate for the development of a cytostatic/cytotoxic drug in the therapy of cancer or other proliferative diseases (DePinto et al., Mol. Cancer Ther. 5: 2644-58, 2006; Joshi et al., Mol. Cancer Ther. 6: 918-25, 2007).
However, while CDK inhibitory activities, e.g., as determined in biochemical kinase inhibition assays in vitro, are highly important parameters for hit and lead identification in research, a successful development of a drug for therapeutic applications will finally depend on many additional factors, such as the in vitro ADMET profile (including solubility and permeability), cell biology studies (including cellular inhibition of disease-related cell lines), pharmacokinetic (including bioavailability) and pharmacodynamic studies (including, most importantly, activity in disease-related animal models), and the toxicological profile.
It is known that certain pyrazolo[3,4-d]pyrimidines, substituted in a specific manner, have pharmacologically useful properties. In particular, certain derivatives of pyrazolo[3,4-d]pyrimidin-4-ones are known to possess anti-proliferative activity (see, Rossi et al., Comput. Aided Mol. Des. 19: 111-22, 2005; Markwalder et al., J. Med. Chem. 47: 5894-911, 2004).
PCT publication WO 00/021926 broadly discloses a class of pyrazolo[3,4-d]pyrimidin-4-ones, including generic structures representing certain 1-phenyl- and 1-pyridyl-pyrazolo[3,4-d]pyrimidin-4-ones. However, no specific 1-pyridyl-substituted compounds are disclosed or their synthesis described, and no data are presented to indicate whether such compounds would exhibit inhibitory properties in, for example, CDK enzymatic assays. There is thus no particularized teaching to select pyridine substituents for producing pharmaceutically useful compounds, and, indeed, no demonstration that 1-pyridyl forms would be as effective as the compounds actually synthesized or effective at all in the disclosed uses. In particular, there is no teaching of the specific compound 1-(3,5-dichloropyridin-4-yl)-6-(3-hydroxy-4-pyrrolidinomethyl-phenyl)methyl-3-isopropyl-pyrazolo[3,4-d]pyrimidin-4-one (Compound I), disclosed below.
PCT publication WO 03/033499 specifically discloses certain 1-phenyl-3-isopropyl-6-arylmethyl-pyrazolo[3,4-d]pyrimidin-4-ones that are shown to inhibit cyclin-dependent kinases and to have certain activity in tumor models (see also WO 2004/092139; WO 2005/063765; see also, Caligiuri et al., Chem Biol. 12: 1103-15, 2005). Of note however, is that these applications do not specially disclose, generically claim or suggest a utility for any corresponding 1-pyridyl-pyrazolo[3,4-d]pyrimidin-4-ones. WO 2004/092139 and WO 2005/063765 both disclose 1-(2,6-dichlorophenyl)-6-(3-hydroxy-4-pyrrolidinomethyl-phenyl)methyl-3-isopropyl-pyrazolo[3,4-d]pyrimidin-4-one (P)

that can be shown to have balanced CDK inhibitory activities in biochemical kinase inhibition assays in vitro against CDK1, CDK2, CDK4, CDK6, CDK7 and CDK9. However, that compound did not have satisfactory properties in terms of solubility, permeability and particularly in xenograft tumor models in nude mice, so that further preclinical or clinical development for the treatment of cancer would not have been reasonably expected.
Thus, despite the progress that has been made, the search continues for low molecular weight kinase inhibitor compounds with balanced CDK inhibitory activities in biochemical kinase inhibition assays in vitro against CDK1, CDK2, CDK4, CDK6, CDK7 and CDK9, that show potent activity in a xenograft tumor model, and that are useful for treating cancer. Such compounds may additionally be useful for treating a wide variety of diseases, including cancer, tumors and other proliferative disorders or diseases including restenosis, angiogenesis, diabetic retinopathy, psoriasis, surgical adhesions, macular degeneration, and atherosclerosis. Thus, a strong need exists to provide compositions, pharmaceuticals and/or medicaments with kinase inhibitor activity or anti-proliferative activity against cells such as tumor cells. Such compositions, pharmaceuticals and/or medicaments may possess not only such activities, but may also exert tolerable, acceptable or diminished side effects in comparison to other kinase inhibitors or anti-proliferative agents. Furthermore, the spectrum of tumor types or other diseases responsive to treatment with such compositions, pharmaceuticals and/or medicaments may be broad. The active ingredients of such compositions, pharmaceuticals and/or medicaments may be suitable for use in the treatment of the mentioned indications as single agent, and/or in combination therapy, be it in connection with other therapeutic agents, with radiation, with operative/surgical procedures, thermal therapy or any other treatment known in the mentioned indications.